First record of Stigmatomyces (Ascomycota: Laboulbeniales) on Drosophilidae from Japan

ABSTRACT Three Stigmatomyces species were detected on five drosophilid species from Japan. We report Stigmatomyces majewskii on Drosophila rufa and Drosophila suzukii, Stigmatomyces scaptodrosophilae on Scaptodrosophila coracina and Scaptodrosophila subtilis, and Stigmatomyces sacaptomyzae on Scaptomyza graminum. Except for Scaptomyza graminum, each of these species is a newly identified Stigmatomyces host. Our discovery that D. suzukii is a host of S. majewskii may provide new pest management approaches for this global agricultural pest insect.


Introduction
Laboulbeniales is an order of small fungi found only on the cuticles of living arthropods. Their complete nutritional dependence from their host (obligate parasitism) differentiates them from the typical Ascomycota [1,2]. The genus Stigmatomyces (family Laboulbeniaceae) consists of ~200 species that are primarily found on Diptera and less frequently on Coleoptera (Staphylinidae, Coccinellidae, and Elateridae) and Hemiptera (Anthocoridae), therefore called 'fly laboulbeniomycetes'. Though 10 Stigmatomyces species hosted by Drosophilidae have been described (Table 1), none have been reported from Japan. One of the authors (MO) discovered a Stigmatomyces species on Drosophila suzukii (Matsumura, 1931) in 2018, and we report on Stigmatomyces collections carried out in Japan between 2019 and 2022.
Our discovery of a frequent Stigmatomyces infection on wild Japanese D. suzukii has an impact on agricultural pest management. D. suzukii is endemic to East and Southeast Asia, but began to rapidly expand globally in the late 2000s [3][4][5]. Females have large serrated ovipositors (hypogynium) that cause extensive damage to ripening fruits and, as a result, is considered a costly invasive agricultural pest [3][4][5]. Research into management strategies for D. suzukii is ongoing [6] and includes investigating the use of pathogenic fungi [7]. Our discovery of a potential natural enemy of D. suzukii potentially opens new management strategies.

Determination of Stigmatomyces on Drosophilidae
We collected Drosophilidae from nine locations in Japan (Fig. S1) between 2019 and 2022, with most samples coming from Fushimi (Kyoto), Sugadaira (Nagano), and Tsukuba (Ibaraki). Flies were caught primarily using bait traps containing banana and yeast. We also collected by sweeping nets over fallen fruits. Flies were anaesthetized with triethylamine and preserved in 70% ethanol before being sorted under a binocular stereo microscope (SMZ-U, Nikon, Tokyo, Japan). The different parts of individual flies were examined to determine whether Stigmatomyces is infected, and the infection frequency on each body part was recorded. To identify the Stigmatomyces species, thalli were removed from the flies using forceps and fixed in 99.9% lactate. They were mounted on glass slides and examined under a light microscope (CX23, Olympus, Tokyo, Japan) or a phase-contrast microscope (BH-2, Olympus). Images were acquired by using a single-lens reflex camera (EOS60D, Canon, Tokyo, Japan) connected with a dedicated adaptor (NY1S, Canon). The sizes of specimens were measured by using 11φ24 and NOB1 micrometres (Fuji Kogaku, Kumamoto, Japan). Scale bar was inserted to the images by using ImageJ 1.53k (https://imagej.nih. gov/ij/). We used R 4.2.1 (https://www.r-project.org) for the statistical tests.

Observation of grooming behaviour of D. suzukii
The behavioural observations used an iso-female strain of D. suzukii (SGD001) that was collected in the Sugadaira highlands (Ueda, Nagano Prefecture) in 2017 and maintained at 25°C under a light/dark cycle (L:D = 14 h:10 h). Virgin flies used for the observations were collected without anaesthesia within 8 h of emergence and kept in male-or female-only groups prior to use. They were individually aspirated to a Petri dish (diameter, 3.6 cm; height, 1.0 cm) and observed at room temperature for 10 min under the binocular stereo microscope. Four age groups were used (4,8,12, and 16 days after emergence), each replicated 10 times (n = 40 for each sex). Because there was no difference in grooming behaviour among the age groups of the same sex, the data were pooled. S. majewskii-infected D. suzukii males were collected from the wild and maintained at 18°C under constant light before use and examined under the same conditions as above (n = 17).

Stigmatomyces majewskii H. L. Dainat, Manier & Balazuc, 1974
We collected D. suzukii flies infected with Stigmatomyces from eight localities of Japan ( Figures. 1(a) and S1). A D. rufa Kikkawa & Peng, 1938 male that was collected from Kyoto on 22 October 2022 was also found to be infected with the same Stigmatomyces species on tibia of his left foreleg (Figure 1(b)). Based on their diagnostic characters [8][9][10][11][12], they were identified as Stigmatomyces majewskii (Figure 1(f),(g)). S. majewskii has a perithecial neck as long as the venter, transparent appendages, and an appendage axis composed of four cells (differentiating it from the axis of Stigmatomyces entomophilus (Peck) Thax., 1900 that is composed of six cells). See Appendix A for the difference between S. majewskii and its close relatives. The length of thalli was 410-590 μm (n = 13).
Although the number of our primary collection sites was limited, our emphasis was placed on identifying infected D. suzukii at every locality (Fig. S1). The collection at Fukuoka (24-29 September 2021) is an exception: no infected D. suzukii were recorded from 26 females and 199 males. Interestingly, our D. suzukii collection (September 2019-October 2022) always exhibited a biased sex ratio (χ 2 = 976.31, d.f. = 1, p < 2.2E-16), with 89.0% (n = 2734) being males. Furthermore, the frequency of infection was significantly higher (χ 2 = 35.495, d.f. = 1, p = 2.557E-9) in males (16.5%, n = 2432) than females (3.3%, n = 302). We observed the frequency of infection to fluctuate seasonally (Appendix B). D. suzukii displayed two seasonal morphs, a summer morph with a lighter body colour and a winter morph with a darker body colour [13]. Both morphs were found infected with S. majewskii (Appendix B). Why did our collection include more D. suzukii males (frequently Stigmatomyses-infected males) than females? We propose five hypotheses for this observation: 1) Our collection method (primarily banana-yeast bait traps) may have been more attractive to males (especially for active, infected males) than females; females may prefer odours from ripening fruits. 2) Males may have the tendency to get infected more easily than females due to unknown sexually dimorphic immune factors or due to higher male-male transmission resulting from higher male-to-male contact compared to female-female contact. 3) There could be an increased nutritional content that the males provide to S. majewskii compared to females. 4) Females may have a higher efficiency in removing S. majewskii compared to males. 5) Infected females may have lower fitness in the field and thus are less numerous. To determine which hypothesis is correct, we are analysing the mechanisms of S. majewskii infection in D. suzukii. Further analyses will be reported in a separate paper.
The S. majewskii infection on D. suzukii males (n = 320) was frequently observed on their forelegs (63.4%; especially on femur (50.0%), tibia (24.7%)), midlegs (44.4%), anterior head (31.3%; such as, maxillary palp, frons, labella, antenna, arista and edge of the compound eyes), and dorsal abdomen (31.3%) ( Figures.  2(a) and S2(a)). On D. suzukii females (n = 7), the infections tend to be located on the anterior of their heads (57.1%), forelegs (42.9%), hindlegs (28.6%), lateral or posterior head (28.6%), and dorsal abdomen (28.6%) ( Figures. 2(b) and S2(b)). In both sexes, the infection was seen on the marginal and proximal regions of the wings and on the left or right positions of the dorsal abdomen (rarely on the mid position). The infection was not observed in the compound eyes, although immature thalli were detected. S. majewskii can infect the entire body of D. suzukii but are probably removed by using their legs (Appendix C). This differs from guava fruit flies (Anasterepha striata Schiner, 1868), where the Stigmatomyces (S. aciurae Thaxt., 1917 and S. verruculosus Thaxt., 1917) infection is restricted to the areas of sexual contact [14]. We arranged our observations of S. majewskii according to their developmental stage (Appendix D). Interestingly, we collected preliminary data that suggests that S. majewskii may include dioecious individuals (Appendix E).
We identified the species to be S. scaptomyzae based on its diagnostic characters: the axis of the appendage is composed of five cells, the stalk-cell of the appendage is broader than the brown and orthogonal basal cell, and the perithecium is brown. The length of thalli was 220-260 μm (n = 5). On Scaptomyza graminum males (n = 9), the infection was frequently seen on legs and abdomen, especially at the ventral side ( Figures. 2(c) and S2(c)). On the females (n = 4), the infection was restricted to hindlegs, genitalia, and abdomen, especially at the dorsal side ( Figures. 2(d) and S2 (d)). Therefore, we suspect that the infection was partly caused by intersexual contact. Furthermore, we have preliminary data suggesting that S. scaptomyzae may include dioecious individuals (Appendix E).

Stigmatomyces scaptodrosophilae W. Rossi & E. Christian, 2020
Stigmatomyces scaptodrosophilae was recently described based on samples found on Scaptodrosophila deflexa (Duda, 1924) from Ukraine and Austria and a third from Kenya on Scaptodrosophila sp [19]. We discovered this species in Fushimi, Kyoto on Scaptodrosophila coracina (Kikkawa & Peng, 1938) and Scaptodrosophila subtilis (Kikkawa & Peng, 1938) and in the Sugadaira highlands, Nagano on Scaptodrosophila coracina (Figure 1(d),(e),(i),(j)). We verified that the axis of the appendage is composed of five to six cells, the stalk-cell of the appendage is broader than the orthogonal basal cell, and the stalk-cell of the perithecium is shorter on the inner side than the stalk-cell of the appendage while being separated from the secondary stalk-cell by an obliquely hollow septum. The length of thalli on Scaptodrosophila coracina was 410-450 μm (n = 3) and was 310-330 μm on Scaptodrosophila subtilis (n = 4). The cause of the different thalli sizes remains unclear but may be related to genetic differences of S. scaptodrosophilae or the nutritional differences provided by the two hosts.

Concluding remarks
In the present report, we identified three Stigmatomyces species on five Japanese drosophilid species. Although the three Stigmatomyces species had been described in other countries, most of their hosts reported here (four species) are new discoveries. Because investigations into the natural history of Stigmatomyces in Asia have so far been limited [20], we suspect that more Stigmatomyces species and their hosts remain to be identified. Additional studies like this one will also help us to understand the impact that Stigmatomyces has on the population dynamics of Drosophilidae in nature. Of particular importance are the effects of S. majewskii on the agricultural pest D. suzukii. As D. suzukii is a global invasive species that originated in Asia, it may be free of natural enemies within in its new habitats. The introduction of predators, parasites, parasitoids, and pathogens presents attractive management strategies, and S. majewskii provides a new and potentially important option to investigate further.